1. Field of the Invention
This invention relates to the genetic engineering and recombinant DNA art and, more particularly, to an improved tagging or labeling of fragments with fluorescent, luminescent, or color reflective labels.
2. Description of the Prior Art
The modern history of genetic engineering is generally accepted to have begun in 1973 when refinements in analytic methods uncovered unanticipated complexities and subtleties in the organization, replication, and expression of DNA. Revision of earlier concepts have been dependent upon the use of molecular tools for dissecting, cloning, and amplifying genomes.
The ability to sequence the DNA is one of the most fundamental and important technological advancements in basic research and now in clinical medicine. Most of the current methods of DNA sequence analysis utilize biologically hazardous, short lived radioisotopes such as phosphorus 32 (32P) to tag the DNA in vitro for determining the size and composition of fragments. However, the process of sequencing is labor intensive and time consuming.
In the chemical method of sequence analysis, the terminal nucleotide of a fragment is labeled and then hydrolyzed by base-specific reaction. The enzymatic replication method of sequence analysis incorporates the cloning of the gene that is to be sequenced and then attaching it to a single stranded DNA bacteriophage. A primer DNA which has the complementary base sequence to the DNA segment adjacent to the cloned gene is used to initiate the copying of the cloned DNA. The process of this synthesis requires four kinds of deoxyribonucleoside triphosphates (dATP, dCTP, dGTP and dTTP) as precursers and four different dideoxyribonucleoside triphosphates (ddATP, ddCTP, ddGTP and ddTTP) as chain terminators. Reaction mixtures for copying the DNA containing all four precursers and only one each of the four chain terminators are placed in four separate tubes. When a dideoxytriphosphate is incorporated to the copied DNA, the subsequent DNA synthesis ceases immediately and chain termination occurs. As a result, all the copied DNA with ddATP, for example, terminates at positions where dATP is normally incorporated. Since incorporations of dideoxytriphosphates occur anywhere in the copied DNA, various lengths of DNA, with dideoxytriphosphate bases at their termini are generated. The copied DNA fragments are then separated by polyacrylamide gel electro-phoresis according to the number of bases in each fragment of DNA. In order to detect the DNA on the gel, a radioactive tag such as 32P labeled deoxyribonucleoside triphosphate is incorporated into the copied DNA. The radioactive DNA is separated on the gel and visualized by exposing the gel to x-ray film. The sequence of the copied DNA is read from the autoradiograph of the gel.
Others have attempted to substitute elements such as fluorescent or luminescent tagging for radioactive tagging. A method developed by Smith, Fung and others The synthesis of oligonucleotides containing an aliphatic amino group at the 5' terminus: synthesis of fluorescent DNA primers for use in DNA sequence analysis, Nucleic Acids Research, Volume 13, Number 7, 1985, page 2399. utilizes chemically synthesized DNA primers with a covalently attached fluorophores as a substitute for the radiolabeled nucleoside triphosphate for use in DNA sequence analysis. However, each fluorescent compound utilized must fluoresce at a significantly different wave length from the other three compounds in order for the user to observe the compound separate and apart from the others. Therefore, four different compounds must be used. The chemical characteristics of each compound is sufficiently different to alter the mobility of the primers in a polyacrylamide gel electro-phoresis. This alteration is readily apparent when attempting to read the pattern of bands after separation. The normal separation of fragments by one base pair in each lane of the gel is compromised by this delta-difference in chemical characteristics of the fluorescent compounds by displacing the band by more than the normal band width for a base pair. Thus the accuracy of determination of the DNA sequence is compromised.
Thus, there has long been a need for a non-radiological tagging which could incorporate compensation for the chemical characteristics of the tagging.
Further, it is also desired that the tagging utilize only a single lane in polyacrylamide gel electro-phoresis in order to allow simplified automatic analysis of sequencing.
Further, it is also desired that the tagging be useful on natural as well as synthesized DNA primers.